Nucleic acid encoding human GPR14 receptor

ABSTRACT

Human GPR14 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing Human GPR14 polypeptides and polynucleotides in the design of protocols for the treatment of infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers; anorexia; bulimia; asthma; Parlinson&#39;s disease; acute heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma; allergies; benign prostatic hypertrophy; and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington&#39;s disease or Gilles dela Tourett&#39;s syndrome, among others and diagnostic assays for such conditions.

FIELD OF INVENTION

This invention relates to newly identified polynucleotides, polypeptidesencoded by them and to the use of such polynucleotides and polypeptides,and to their production. More particularly, the polynucleotides andpolypeptides of the present invention relate to G-Protein coupledreceptor, hereinafter referred to as Human GPR14. The invention alsorelates to inhibiting or activating the action of such polynucleotidesand polypeptides.

BACKGROUND OF THE INVENTION

It is well established that many medically significant biologicalprocesses are mediated by proteins participating in signal transductionpathways that involve G-proteins and/or second messengers, e.g., cAMP(Lefkowitz, Nature, 1991, 351:353-354). Herein these proteins arereferred to as proteins participating in pathways with G-proteins or PPGproteins. Some examples of these proteins include the GPC receptors,such as those for adrenergic agents and dopamine (Kobilka, B. K., etal., Proc. Natl Acad. Sci., USA, 1987, 84:46-50; Kobilka, B. K., et al.,Science, 1987, 238:650-656; Bunzow, J. R., et al., Nature, 1988,336:783-787), G-proteins themselves, effector proteins, e.g.,phospholipase C, adenyl cyclase, and phosphodiesterase, and actuatorproteins, e.g., protein kinase A and protein kinase C (Simon, M. I., etal., Science, 1991, 252:802-8).

For example, in one form of signal transduction, the effect of hormonebinding is activation of the enzyme, adenylate cyclase, inside the cell.Enzyme activation by hormones is dependent on the presence of thenucleotide GTP. GTP also influences hormone binding. A G-proteinconnects the hormone receptor to adenylate cyclase. G-protein was shownto exchange GTP for bound GDP when activated by a hormone receptor. TheGTP-carrying form then binds to activated adenylate cyclase. Hydrolysisof GTP to GDP, catalyzed by the G-protein itself, returns the G-proteinto its basal, inactive form. Thus, the G-protein serves a dual role, asan intermediate that relays the signal from receptor to effector, and asa clock that controls the duration of the signal.

The membrane protein gene superfamily of G-protein coupled receptors hasbeen characterized as having seven putative transmembrane domains. Thedomains are believed to represent transmembrane a-helices connected byextracellular or cytoplasmic. loops. G-protein coupled receptors includea wide range of biologically active receptors, such as hormone, viral,growth factor and neuroreceptors.

G-protein coupled receptors have been characterized as including theseseven conserved hydrophobic stretches of about 20 to 30 amino acids,connecting at least eight divergent hydrophilic loops. The G-proteinfamily of coupled receptors includes dopamine receptors which bind toneuroleptic drugs used for treating psychotic and neurologicaldisorders. Other examples of members of this family include, but are notlimited to, calcitonin, adrenergic, endothelin, cAMP, adenosine,muscarinic, acetylcholine, serotonin, histamine, thrombin, kinin,follicle stimulating hormone, opsins, endothelial differentiationgene-1, rhodopsins, odorant, and cytomegalovirus receptors.

Most G-protein coupled receptors (or otherwise known as 7TM receptors)have single conserved cysteine residues in each of the first twoextracellular loops which form disulfide bonds that are believed tostabilize functional protein structure. The 7 transmembrane regions aredesignated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 has beenimplicated in signal transduction.

Phosphorylation and lipidation (palmitylation or famesylation) ofcysteine residues can influence signal transduction of some G-proteincoupled receptors. Most G-protein coupled receptors contain potentialphosphorylation sites within the third cytoplasmic loop and/or thecarboxy terminus. For several G-protein coupled receptors, such as theb-adrenoreceptor, phosphorylation by protein kinase A and/or specificreceptor kinases mediates receptor desensitization.

For some receptors, the ligand binding sites of G-protein coupledreceptors are believed to comprise hydrophilic sockets formed by severalG-protein coupled receptor transmembrane domains, said socket beingsurrounded by hydrophobic residues of the G-protein coupled receptors.The hydrophilic side of each G-protein coupled receptor transmembranehelix is postulated to face inward and form polar ligand binding site.TM3 has been implicated in several G-protein coupled receptors as havinga ligand binding site, such as the TM3 aspartate residue. TM5 serines, aTM6 asparagine and TM6 or TM7 phenylalanines or tyrosines are alsoimplicated in ligand binding.

G-protein coupled receptors can be intracellularly coupled byheterotrimeric G-proteins to various intracellular enzymes, ion channelsand transporters (see, Johnson et al., Endoc. Rev., 1989, 10:317-331)Different G-protein a-subunits preferentially stimulate particulareffectors to modulate various biological functions in a cell.Phosphorylation of cytoplasmic residues of G-protein coupled receptorshave been identified as an important mechanism for the regulation ofG-protein coupling of some G-protein coupled receptors. G-proteincoupled receptors are found in numerous sites within a mammalian host.

Over the past 15 years, nearly 350 therapeutic agents targeting 7transmembrane (7 TM) receptors have been successfully introduced ontothe market.

This indicates that these receptors have an established, proven historyas therapeutic targets. Clearly there is a need for identification andcharacterization of further receptors which can play a role inpreventing, ameliorating or correcting dysfunctions or diseases,including, but not limited to, infections such as bacterial, fungal,protozoan and viral infections, particularly infections caused by HIV-1or HIV-2; pain; cancers; anorexia; bulimia; asthma; Parkinson's disease;acute heart failure; hypotension; hypertension; urinary retention;osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma;allergies; benign prostatic hypertrophy; and psychotic and neurologicaldisorders, including anxiety, schizophrenia, manic depression, delirium,dementia, severe mental retardation and dyskinesias, such asHuntington's disease or Gilles dela Tourett's syndrome.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to Human GPR14 polypeptides andrecombinant materials and methods for their production. Another aspectof the invention relates to methods for using such Human GPR14polypeptides and polynucleotides. Such uses include the treatment ofinfections such as bacterial, fungal, protozoan and viral infections,particularly infections caused by HIV-1 or HIV-2; pain; cancers;anorexia; bulimia; asthma; Parkinson's disease; acute heart failure;hypotension; hypertension; urinary retention; osteoporosis; anginapectoris; myocardial infarction; ulcers; asthma; allergies; benignprostatic hypertrophy; and psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, delirium, dementia,severe mental retardation and dyskinesias, such as Huntington's diseaseor Gilles dela Tourett's syndrome, among others. In still anotheraspect, the invention relates to methods to identify agonists andantagonists using the materials provided by the invention, and treatingconditions associated with Human GPR14 imbalance with the identifiedcompounds. Yet another aspect of the invention relates to diagnosticassays for detecting diseases associated with inappropriate Human GPR14activity or levels.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 1C show the nucleotide and deduced amino acid sequenceof Human GPR 14. SEQ ID NOS: 1 and 2.

DESCRIPTION OF THE INVENTION Definitions

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

"Human GPR14" refers generally to a polypeptide having the amino acidsequence set forth in SEQ ID NO:2, or an allelic variant thereof.

"Receptor Activity" or "Biological Activity of the Receptor" refers tothe metabolic or physiologic function of said Human GPR14 includingsimilar activities or improved activities or these activities withdecreased undesirable side-effects. Also included are antigenic andimmunogenic activities of said Human GPR14.

"Human GPR14 polypeptides" refers to polypeptides with amino acidsequences sufficiently similar to Human GPR14 sequences, preferablyexhibiting at least one biological activity of the receptor.

"Human GPR14 gene" refers to a polynucleotide having the nucleotidesequence set forth in SEQ ID NO: 1 or allelic variants thereof and/ortheir complements.

"Human GPR14 polynucleotides" refers to polynucleotides containing anucleotide sequence which encodes a Human GPR14 polypeptide or fragmentthereof, or a nucleotide sequence which has at least 75.9% identity to anucleotide sequence encoding the polypeptide of SEQ ID NO:2 or thecorresponding fragment thereof, or a nucleotide sequence which hassufficient identity to a nucleotide sequence contained in SEQ ID NO:1 tohybridize under conditions useable for amplification or for use as aprobe or marker.

"Antibodies" as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of an Fab or other immunoglobulinexpression library.

"Isolated" means altered "by the hand of man" from the natural state. Ifan "isolated" composition or substance occurs in nature, it has beenchanged or removed from its original environment, or both. For example,a polynucleotide or a polypeptide naturally present in a living animalis not "isolated," but the same polynucleotide or polypeptide separatedfrom the coexisting materials of its natural state is "isolated", as theterm is employed herein.

"Polynucleotide" generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. "Polynucleotides" include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, "polynucleotide" refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. "Modified" basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications has been made to DNA and RNA; thus,"polynucleotide" embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. "Polynucleotide" also embraces relatively short polynucleotides,often referred to as oligonucleotides.

"Polypeptide" refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. "Polypeptide" refers to both shortchains, commonly referred to as peptides, oligopeptides or oligomers,and to longer chains, generally referred to as proteins. Polypeptidesmay contain amino acids other than the 20 gene-encoded amino acids."Polypeptides" include amino acid sequences modified either by naturalprocesses, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from posttranslation natural processes ormay be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination. See, for instance, PROTEINS--STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork, 1993 and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York,1983; Seifter et al., "Analysis for protein modifications and nonproteincofactors", Meth Enzymol (1990) 182:626-646 and Rattan et al., "ProteinSynthesis: Posttranslational Modifications and Aging", Ann NY Acad Sci(1992) 663:48-62.

"Variant" as the term is used herein, is a polynucleotide or polypeptidethat differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniquesor by direct synthesis.

"Identity" is a measure of the identity of nucleotide sequences or aminoacid sequences. In general, the sequences are aligned so that thehighest order match is obtained. "Identity" per se has an art-recognizedmeaning and can be calculated using published techniques. See, e.g.:(COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS,Smith, D. W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OFSEQUENCE DATA, PART I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, vonHeinje, G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).While there exist a number of methods to measure identity between twopolynucleotide or polypeptide sequences, the term "identity" is wellknown to skilled artisans (Carillo, H., and Lipton, D., SIAM J AppliedMath (1988) 48:1073). Methods commonly employed to determine identity orsimilarity between two sequences include, but are not limited to, thosedisclosed in Guide to Huge Computers, Martin J. Bishop, ed., AcademicPress, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J AppliedMath (1988) 48:1073. Methods to determine identity and similarity arecodified in computer programs. Preferred computer program methods todetermine identity and similarity between two sequences include, but arenot limited to, GCS program package (Devereux, J., et al., Nucleic AcidsResearch (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S. F. etal., J Molec Biol (1990) 215:403).

Polypeptides of the Invention

The Human GPR14 polypeptides of the present invention include thepolypeptide of SEQ ID NO:2 (in particular the mature polypeptide) aswell as Human GPR14 polypeptides and which have at least 84.25% identityto the polypeptide of SEQ ID NO:2 or the relevant portion and morepreferably at least 85% identity, and still more preferably at least 90%identity, and even still more preferably at least 95% identity to SEQ IDNO: 2.

The Human GPR14 polypeptides may be in the form of the "mature" proteinor may be a part of a larger protein such as a fusion protein. It isoften advantageous to include an additional amino acid sequence whichcontains secretory or leader sequences, pro-sequences, sequences whichaid in purification such as multiple histidine residues, or anadditional sequence for stability during recombinant production.

Biologically active fragments of the Human GPR14 polypeptides are alsoincluded in the invention. A fragment is a polypeptide having an aminoacid sequence that entirely is the same as part, but not all, of theamino acid sequence of the aforementioned Human GPR 14 polypeptides. Aswith Human GPR14 polypeptides, fragments may be "free-standing," orcomprised within a larger polypeptide of which they form a part orregion, most preferably as a single continuous region. Representativeexamples of polypeptide fragments of the invention, include, forexample, fragments from about amino acid number 1-20, 21-40, 41-60,61-80, 81-100, and 101 to the end of Human GPR14 polypeptide. In thiscontext "about" includes the particularly recited ranges larger orsmaller by several, 5, 4, 3, 2 or 1 amino acid at either extreme or atboth extremes.

Preferred fragments include, for example, truncation polypeptides havingthe amino acid sequence of Human GPR14 polypeptides, except for deletionof a continuous series of residues that includes the amino terminus, ora continuous series of residues that includes the carboxyl terminus ordeletion of two continuous series of residues, one including the aminoterminus and one including the carboxyl terminus. Also preferred arefragments characterized by structural or functional attributes such asfragments that comprise alpha-helix and alpha-helix forming regions,beta-sheet and beta-sheet-forming regions, turn and turn-formingregions, coil and coil-forming regions, hydrophilic regions, hydrophobicregions, alpha amphipathic regions, beta amphipathic regions, flexibleregions, surface-forming regions, substrate binding region, and highantigenic index regions. Biologically active fragments are those thatmediate receptor activity, including those with a similar activity or animproved activity, or with a decreased undesirable activity. Alsoincluded are those that are antigenic or immunogenic in an animal,especially in a human.

Thus, the polypeptides of the invention include polypeptides having anamino acid sequence at least 84.25% identical to that of SEQ ID NO:2 orfragments thereof with at least 84.25% identity to the correspondingfragment of SEQ ID NO:2. Preferably, all of these polypeptides retainthe biological activity of the receptor, including antigenic activity.Included in this group are variants of the defined sequence andfragments. Preferred variants are those that vary from the referents byconservative amino acid substitutions--i.e., those that substitute aresidue with another of like characteristics. Typical such substitutionsare among Ala, Val, Leu and Ile; among Ser and Thr; among the acidicresidues Asp and Glu; among Asn and Gln; and among the basic residuesLys and Arg: or aromatic residues Phe and Tyr. Particularly preferredare variants in which several, 5-10, 1-5, or 1-2 amino acids aresubstituted, deleted, or added in any combination.

The Human GPR14 polypeptides of the invention can be prepared in anysuitable manner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

Polynucleotides of the Invention

Another aspect of the invention relates to isolated polynucleotideswhich encode the Human GPR14 polypeptides and polynucleotides closelyrelated thereto.

Human GPR14 of the invention is structurally related to other proteinsof the G-Protein coupled receptor, as shown by the results of sequencingthe cDNA encoding Human GPR14. The cDNA sequence contains an openreading frame encoding a protein of 390 with a deduced molecular weightof kDa. Human GPR14 of FIGS. 1A, 1B, and 1C (SEQ ID NO:2) has about84.25% identity (using Fasta) in 387 amino acid residues with Rattusnorvegicus GPR14 orphan receptor (A. Marches, et al., Genomics29(2):335-344, 1995). Furthermore, human GPR14 (SEQ. ID NO:2) is 31.7%identical to human Somatostatin-3 receptor over 331 amino acid residues(Y. Yamada et al., Mol. Endocrinol. 6(12):2136-2142, 1992). Human GPR14gene of FIGS. 1A, 1B, and 1C (SEQ ID NO:1) has about 75.9% identity(using blast) in 1539 bp nucleotide residues with Rattus norvegicusGPR14 orphan receptor (A. Marches, et al., Genomics 29(2):335-344,1995). Furthermore, human GGPR 14(SEQ. ID NO: 1) is 79% identical over783bp to Rattus norvegicus G- protein couple receptor SENR (M. Tal, etal., Biochem. Biophys. Res. Commun. 209(2):752-759 1995.)

One polynucleotide of the present invention encoding Human GPR14 may beobtained using standard cloning and screening, from a cDNA libraryderived from mRNA in cells of human placenta using the expressedsequence tag (EST) analysis (Adams, M. D., et al. Science (1991)252:1651-1656; Adams, M. D. et al., Nature, (1992) 355:632-634; Adams,M. D., et al., Nature (1995) 377 Supp:3-174). Polynucleotides of theinvention can also be obtained from natural sources such as genomic DNAlibraries or can be synthesized using well known and commerciallyavailable techniques.

Thus, the nucleotide sequence encoding Human GPR14 polypeptides may beidentical over its entire length to the coding sequence in FIGS. 1A, 1B,and 1C (SEQ ID NO: 1), or may be a degenerate form of this nucleotidesequence encoding the polypeptide of SEQ ID NO:2, or may be highlyidentical to a nucleotide sequence that encodes the polypeptide of SEQID NO:2. Preferably, the polynucleotides of the invention contain anucleotide sequence that is highly identical, at least 75.9% identical,with a nucleotide sequence encoding a Human GPR14 polypeptide, or atleast 75.9% identical with the encoding nucleotide sequence set forth inFIGS. 1A, 1B, and 1C (SEQ ID NO: 1), or at least 75.9% identical to anucleotide sequence encoding the polypeptide of SEQ ID NO:2.

When the polynucleotides of the invention are used for the recombinantproduction of Human GPR14 polypeptide, the polynucleotide may includethe coding sequence for the mature polypeptide or a fragment thereof, byitself; the coding sequence for the mature polypeptide or fragment inreading frame with other coding sequences, such as those encoding aleader or secretory sequence, a pre-, or pro- or prepro- proteinsequence, or other fusion peptide portions. For example, a markersequence which facilitates purification of the fused polypeptide can beencoded. In certain preferred embodiments of this aspect of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., ProcNatl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotideray also contain non-coding 5' and 3' sequences, such as transcribed,non-translated sequences, splicing and polyadenylation signals, ribosomebinding sites and sequences that stabilize mRNA.

Among particularly preferred embodiments of the invention arepolynucleotides encoding Human GPR14 polypeptides having the amino acidsequence of set out in FIGS. 1A, 1B, and 1C (SEQ ID NO:2) and variantsthereof.

Further preferred embodiments are polynucleotides encoding Human GPR14variants that have the amino acid sequence of the Human GPR14 of FIGS.1A, 1B, and 1C (SEQ ID NO:2) in which several, 5-10, 1-5, 1-3, 1-2 or 1amino acid residues are substituted, deleted or added, in anycombination.

Further preferred embodiments of the invention are polynucleotides thatare at least 75.9% identical over their entire length to apolynucleotide encoding the Human GPR14 polypeptide having the aminoacid sequence set out in FIGS. 1A, 1B, and 1C (SEQ ID NO:2), andpolynucleotides which are complementary to such polynucleotides. In thisregard, polynucleotides at least 80% identical over their entire lengthto the same are particularly preferred, and those with at least 90% areespecially preferred. Furthermore, those with at least 97% are highlypreferred and those with at least 98-99% are most highly preferred, withat least 99% being the most preferred.

The present invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the presentinvention especially relates to polynucleotides which hybridize understringent conditions to the herein above-described polynucleotides. Asherein used, the term "stringent conditions" means hybridization willoccur only if there is at least 95% and preferably at least 97% identitybetween the sequences.

Polynucleotides of the invention, which are sufficiently identical to anucleotide sequence contained in SEQ ID NO: 1, may be used ashybridization probes for cDNA and genomic DNA, to isolate full-lengthcDNAs and genomic clones encoding Human GPR14 and to isolate cDNA andgenomic clones of other genes that have a high sequence similarity tothe Human GPR14 gene. Such hybridization techniques are known to thoseof skill in the art. Typically these nucleotide sequences are 70%identical, preferably 80% identical, more preferably 90% identical tothat of the referent. The probes generally will comprise at least 15nucleotides. Preferably, such probes will have at least 30 nucleotidesand may have at least 50 nucleotides. Particularly preferred probes willrange between 30 and 50 nucleotides.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsand diagnostics to animal and human disease.

Vectors, Host Cells, Expression

The present invention also relates to vectors which comprise apolynucleotide or polynucleotides of the present invention, and hostcells which are genetically engineered with vectors of the invention andto the production of polypeptides of the invention by recombinanttechniques. Cell-free translation systems can also be employed toproduce such proteins using RNAs derived from the DNA constructs of thepresent invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof for polynucleotidesof the present invention. Introduction of polynucleotides into hostcells can be effected by methods described in many standard laboratorymanuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY (1986)and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)such as calcium phosphate transfection, DEAE-dextran mediatedtransfection, transvection, microinjection, cationic lipid-mediatedtransfection, electroporation, transduction, scrape loading, ballisticintroduction or infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, E. coli, Streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanomacells; and plant cells.

A great variety of expression systems can be used. Such systems include,among others, chromosomal, episoral and virus-derived systems, e.g.,vectors derived from bacterial plasmids, from bacteriophage, fromtransposons, from yeast episomes, from insertion elements, from yeastchromosomal elements, from viruses such as baculoviruses, papovaviruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses;pseudorabies viruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression systems may contain control regions that regulate as well asengender expression. Generally, any system or vector suitable tomaintain, propagate or express polynucleotides to produce a polypeptidein a host may be used. The appropriate nucleotide sequence may beinserted into an expression system by any of a variety of well-known androutine techniques, such as, for example, those set forth in Sambrook etal., MOLECULAR CLONING, A LABORATORY MANUAL (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the desired polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

If the Human GPR14 polypeptide is to be expressed for use in screeningassays, generally, it is preferred that the polypeptide be produced atthe surface of the cell. In this event, the cells may be harvested priorto use in the screening assay. If Human GPR14 polypeptide is secretedinto the medium, the medium can be recovered in order to recover andpurify the polypeptide; if produced intracellularly, the cells mustfirst be lysed before the polypeptide is recovered.

Human GPR14 polypeptides can be recovered and purified from recombinantcell cultures by well-known methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Most preferably, high performance liquidchromatography is employed for purification. Well known techniques forrefolding proteins may be employed to regenerate active conformationwhen the polypeptide is denatured during isolation and or purification.

Diagnostic Assays

This invention also relates to the use of Human GPR14 polynucleotidesfor use as diagnostic reagents. Detection of a mutated form of HumanGPR14 gene associated with a dysfunction will provide a diagnostic toolthat can add to or define a diagnosis of a disease or susceptibility toa disease which results from under-expression, over-expression oraltered expression of Human GPR14. Individuals carrying mutations in theHuman GPR14 gene may be detected at the DNA level by a variety oftechniques.

Nucleic acids for diagnosis may be obtained from a subject's cells, suchas from blood, urine, saliva, tissue biopsy or autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR or other amplification techniques prior toanalysis. RNA or cDNA may also be used in similar fashion. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to the normal genotype. Point mutations can be identifiedby hybridizing amplified DNA to labeled Human GPR14 nucleotidesequences. Perfectly matched sequences can be distinguished frommismatched duplexes by RNase digestion or by differences in meltingtemperatures. DNA sequence differences may also be detected byalterations in electrophoretic mobility of DNA fragments in gels, withor without denaturing agents, or by direct DNA sequencing. See, e.g.,Myers et al., Science (1985) 230:1242. Sequence changes at specificlocations may also be revealed by nuclease protection assays, such asRNase and S1 protection or the chemical cleavage method. See Cotton eta., Proc Natl Acad Sci USA (1985) 85:4397-4401.

The diagnostic assays offer a process for diagnosing or determining asusceptibility to infections such as bacterial, fungal protozoan andviral infections, particularly infections caused by HIV-1 or HIV-2;pain; cancers; anorexia; bulimia; asthma; Parkinson's disease; acuteheart failure; hypotension; hypertension; urinary retention;osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma;allergies; benign prostatic hypertrophy; and psychotic and neurologicaldisorders, including anxiety, schizophrenia, manic depression, delirium,dementia, severe mental retardation and dyskinesias, such asHuntington's disease or Gilles dela Tourett's syndrome through detectionof mutation in the Human GPR14 gene by the methods described.

In addition, infections such as bacterial, fungal, protozoan and viralinfections, particularly infections caused by HIV-1 or HIV-2; pain;cancers; anorexia; bulimia; asthma; Parkinson's disease; acute heartfailure; hypotension; hypertension; urinary retention; osteoporosis;angina pectoris; myocardial infarction; ulcers; asthma; allergies;benign prostatic hypertrophy; and psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, delirium, dementia,severe mental retardation and dyskinesias, such as Huntington's diseaseor Gilles dela Tourett's syndrome, can be diagnosed by methodscomprising determining from a sample derived from a subject anabnormally decreased or increased level of Human GPR14 polypeptide orHuman GPR14 mRNA. Decreased or increased expression can be measured atthe RNA level using any of the methods well known in the art for thequantitation of polynucleotides, such as, for example, PCR, RT-PCR,RNase protection, Northern blotting and other hybridization methods.Assay techniques that can be used to determine levels of a protein, suchas an Human GPR14, in a sample derived from a host are well-known tothose of skill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

Chromosome Assays

The nucleotide sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. The mapping of relevant sequences to chromosomes accordingto the present invention is an important first step in correlating thosesequences with gene associated disease. Once a sequence has been mappedto a precise chromosomal location, the physical position of the sequenceon the chromosome can be correlated with genetic map data. Such data arefound, for example, in V. McKusick, Mendelian Inheritance in Man(available on line through Johns Hopkins University Welch MedicalLibrary). The relationship between genes and diseases that have beenmapped to the same chromosomal region are then identified throughlinkage analysis (coinheritance of physically adjacent genes).

The differences in the cDNA or genomic sequence between affected andunaffected individuals can also be determined. If a mutation is observedin some or all of the affected individuals but not in any normalindividuals, then the mutation is likely to be the causative agent ofthe disease.

Antibodies

The polypeptides of the invention or their fragments or analogs thereof,or cells expressing them can also be used as immunogens to produceantibodies immunospecific for the Human GPR14 polypeptides. The term"immunospecific" means that the antibodies have substantiall greateraffinity for the polypeptides of the invention than their affinity forother related polypeptides in the prior art.

Antibodies generated against the Human GPR14 polypeptides can beobtained by administering the polypeptides or epitope-bearing fragments,analogs or cells to an animal, preferably a nonhuman, using routineprotocols. For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler, G. and Milstein,C., Nature (1975) 256:495-497), the trioma technique, the human B-cellhybridomna technique (Kozbor et al., Immunology Today (1983) 4:72) andthe EBV-hybridoma technique (Cole et al., MONOCLONAL ANTIBODIES ANDCANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can also be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms including other mammals, may be used to express humanizedantibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptides byaffinity chromatography.

Antibodies against Human GPR14 polypeptides may also be employed totreat infections such as bacterial, fungal, protozoan and viralinfections, particularly infections caused by HIV-1 or HIV-2; pain;cancers; anorexia; bulimia; asthma; Parkinson's disease; acute heartfailure; hypotension; hypertension; urinary retention; osteoporosis;angina pectoris; myocardial infarction; ulcers; asthma; allergies;benign prostatic hypertrophy; and psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, delirium, dementia,severe mental retardation and dyskinesias, such as Huntington's diseaseor Gilles dela Tourett's syndrome, among others.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in a mammal which comprises inoculating themammal with Human GPR14 polypeptide, or a fragment thereof, adequate toproduce antibody and/or T cell immune response to protect said animalfrom infections such as bacterial, fungal, protozoan and viralinfections, particularly infections caused by HIV-1 or HIV-2; pain;cancers; anorexia; bulimia; asthma; Parkinson's disease; acute heartfailure; hypotension; hypertension; urinary retention; osteoporosis;angina pectoris; myocardial infarction; ulcers; asthma; allergies;benign prostatic hypertrophy; and psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, delirium, dementia,severe mental retardation and dyskinesias, such as Huntington's diseaseor Gilles dela Tourett's syndrome, among others. Yet another aspect ofthe invention relates to a method of inducing immunological response ina mammal which comprises, delivering Human GPR14 gene via a vectordirecting expression of Human GPR14 polypeptide in vivo in order toinduce such an immunological response to produce antibody to protectsaid animal from diseases.

Further aspect of the invention relates to an immunological/vaccineformulation (composition) which, when introduced into a mammalian host,induces an immunological response in that mammal to a Human GPR14polypeptide wherein the composition comprises a Human GPR14 polypeptideor Human GPR14 gene. The vaccine formulation may further comprise asuitable carrier. Since Human GPR14 polypeptide may be broken down inthe stomach, it is preferably administered parenterally (includingsubcutaneous, intramuscular, intravenous, intradermal etc. injection).Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation instonicwith the blood of the recipient; and aqueous and non-aqueous sterilesuspensions which may include suspending agents or thickening agents.The formulations may be presented in unit-dose or multi-dose containers,for example, sealed ampoules and vials and may be stored in afreeze-dried condition requiring only the addition of the sterile liquidcarrier immediately prior to use. The vaccine formulation may alsoinclude adjuvant systems for enhancing the immunogenicity of theformulation, such as oil-in water systems and other systems known in theart. The dosage will depend on the specific activity of the vaccine andcan be readily determined by routine experimentation.

Screening Assays

The Human GPR14 of the present invention may be employed in a screeningprocess for compounds which bind the receptor and which activate(agonists) or inhibit activation of (antagonists) the receptorpolypeptide of the present invention. Thus, polypeptides of theinvention may also be used to assess the binding of small moleculesubstrates and ligands in, for example, cells, cell-free preparations,chemical libraries, and natural product mixtures. These substrates andligands may be natural substrates and ligands or may be structural orfunctional mimetics. See Coligan et al., Current Protocols in Immunology1(2):Chapter 5 (1991).

Human GPR14 proteins are ubiquitous in the mammalian host and areresponsible for many biological functions, including many pathologies.Accordingly, it is desirous to find compounds and drugs which stimulateHuman GPR14 on the one band and which can inhibit the function of HumanGPR14 on the other hand. In general, agonists are employed fortherapeutic and prophylactic purposes for such conditions as infectionssuch as bacterial, fungal, protozoan and viral infections, particularlyinfections caused by HIV-1 or HIV-2; pain; cancers; anorexia; bulimia;asthma; Parkinson's disease; acute heart failure; hypotension;hypertension; urinary retention; osteoporosis; angina pectoris;myocardial infarction; ulcers; asthma; allergies; benign prostatichypertrophy; and psychotic and neurological disorders, includinganxiety, schizophrenia, manic depression, delirium, dementia, severemental retardation and dyskinesias, such as Huntington's disease orGilles dela Tourett's syndrome. Antagonists may be employed for avariety of therapeutic and prophylactic purposes for such conditions asinfections such as bacterial, fungal, protozoan and viral infections,particularly infections caused by HIV-1 or HIV-2; pain; cancers;anorexia; bulimia; asthma; Parkinson's disease; acute heart failure;hypotension; hypertension; urinary retention; osteoporosis; anginapectoris; myocardial infarction; ulcers; asthma; allergies; benignprostatic hypertrophy; and psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, delirium, dementia,severe mental retardation and dyskinesias, such as Huntington's diseaseor Gilles dela Tourett's syndrome.

In general, such screening procedures involve producing appropriatecells which express the receptor polypeptide of the present invention onthe surface thereof. Such cells include cells from mammals, yeast,Drosophila or E. coli. Cells expressing the receptor (or cell membranecontaining the expressed receptor) are then contacted with a testcompound to observe binding, or stimulation or inhibition of afunctional response.

One screening technique includes the use of cells which express receptorof this invention (for example, transfected CHO cells) in a system whichmeasures extracellular pH or intracellular calcium changes caused byreceptor activation. In this technique, compounds may be contacted withcells expressing the receptor polypeptide of the present invention. Asecond messenger response, e.g., signal transduction, pH changes, orchanges in calcium level, is then measured to determine whether thepotential compound activates or inhibits the receptor.

Another method involves screening for receptor inhibitors by determininginhibition or stimulation of receptor-mediated cAMP and/or adenylatecyclase accumulation. Such a method involves transfecting a eukaryoticcell with the receptor of this invention to express the receptor on thecell surface. The cell is then exposed to potential antagonists in thepresence of the receptor of this invention. The amount of cAMPaccumulation is then measured. If the potential antagonist binds thereceptor, and thus inhibits receptor binding, the levels ofreceptor-mediated cAMP, or adenylate cyclase, activity will be reducedor increased.

Another methods for detecting agonists or antagonists for the receptorof the present invention is the yeast based technology as described inU.S. Pat. No. 5,482,835.

The assays may simply test binding of a candidate compound whereinadherence to the cells bearing the receptor is detected by means of alabel directly or indirectly associated with the candidate compound orin an assay involving competition with a labeled competitor. Further,these assays may test whether the candidate compound results in a signalgenerated by activation of the receptor, using detection systemsappropriate to the cells bearing the receptor at their surfaces.Inhibitors of activation are generally assayed in the presence of aknown agonist and the effect on activation by the agonist by thepresence of the candidate compound is observed. Standard methods forconducting such screening assays are well understood in the art.

Examples of potential Human GPR14 antagonists include antibodies or, insome cases, oligonucleotides or proteins which are closely related tothe ligand of the Human GPR14, e.g., a fragment of the ligand, or smallmolecules which bind to the receptor but do not elicit a response, sothat the activity of the receptor is prevented.

Prophylactic and Therapeutic Methods

This invention provides methods of treating an abnormal conditionsrelated to both an excess of and insufficient amounts of Human GPR14activity.

If the activity of Human GPR14 is in excess, several approaches areavailable. One approach comprises administering to a subject aninhibitor compound (antagonist) as hereinabove described along with apharmaceutically acceptable carrier in an amount effective to inhibitactivation by blocking binding of ligands to the Human GPR14, or byinhibiting a second signal, and thereby alleviating the abnormalcondition.

In another approach, soluble forms of Human GPR14 polypeptides stillcapable of binding the ligand in competition with endogenous Human GPR14may be administered. Typical embodiments of such competitors comprisefragments of the Human GPR 14 polypeptide.

In still another approach, expression of the gene encoding endogenousHuman GPR14 can be inhibited using expression blocking techniques. Knownsuch techniques involve the use of antisense sequences, eitherinternally generated or separately administered. See, for example,O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988). Alternatively, oligonucleotides which form triple helices withthe gene can be supplied. See, for example, Lee et al., Nucleic AcidsRes (1979) 6:3073; Cooney et al., Science (1988) 241:456; Dervan et al.,Science (1991) 251:1360. These oligomers can be administered per se orthe relevant oligomers can be expressed in vivo.

For treating abnormal conditions related to an under-expression of HumanGPR14 and its activity, several approaches are also available. Oneapproach comprises administering to a subject a therapeuticallyeffective amount of a compound which activates Human GPR14, i.e., anagonist as described above, in combination with a pharmaceuticallyacceptable carrier, to thereby alleviate the abnormal condition.Alternatively, gene therapy may be employed to effect the endogenousproduction of Human GPR14 by the relevant cells in the subject. Forexample, a polynucleotide of the invention may be engineered forexpression in a replication defective retroviral vector, as discussedabove. The retroviral expression construct may then be isolated andintroduced into a packaging cell transduced with a retroviral plasmidvector containing RNA encoding a polypeptide of the present inventionsuch that the packaging cell now produces infectious viral particlescontaining the gene of interest. These producer cells may beadministered to a subject for engineering cells in vivo and expressionof the polypeptide in vivo. For overview of gene therapy, see Chapter20, Gene Therapy and other Molecular Genetic-based TherapeuticApproaches, (and references cited therein) in Human Molecular Genetics,T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996).

Formulation and Administration

Peptides, such as the soluble form of Human GPR14 polypeptides, andagonists and antagonist peptides or small molecules, ray be formulatedin combination with a suitable pharmaceutical carrier. Such formulationscomprise a therapeutically effective amount of the polypeptide orcompound, and a pharmaceutically acceptable carrier or excipient. Suchcarriers include but are not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof.Formulation should suit the mode of administration, and is well withinthe skill of the art. The invention further relates to pharmaceuticalpacks and kits comprising one or more containers filled with one or moreof the ingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

Preferred forms of systemic administration of the pharmaceuticalcompositions include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if properly formulated in enteric or encapsulatedformulations, oral administration may also be possible. Administrationof these compounds may also be topical and/or localized, in the form ofsalves, pastes, gels and the like.

The dosage range required depends on the choice of peptide, the route ofadministration, the nature of the formulation, the nature of thesubject's condition, and the judgment of the attending practitioner.Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject.Wide variations in the needed dosage, however, are to be expected inview of the variety of compounds available and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization, as is well understood in the art.

Polypeptides used in treatment can also be generated endogenously in thesubject, in treatment modalities often referred to as "gene therapy" asdescribed above. Thus, for example, cells from a subject may beengineered with a polynucleotide, such as a DNA or RNA, to encode apolypeptide ex vivo, and for example, by the use of a retroviral plasmidvector. The cells are then introduced into the subject.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples illustrate, but do not limitthe invention.

Example 1

A 1.2 kb PCR fragment corresponding to the entire coding region of theRattus norvegicus orphan receptor (GPR14, A. Marchese et al. Genomics,29(2): 335-44, 1995) was used as a probe to screen a total of 0.75Mplaques from a Human Genomic Placenta library (Stratagene, LaJollaCalif., Cat. # 946206). The genomic library screening procedure isdescribed by (Elgin, et al. Stratatgies 4: 8-9, 1991). The probes wereα-32P labeled, using Random Primed Labeling Kit (Boheringer Manheim,Germany, Cat. # 1585584) and purified by running over Sephadex G-50columns (Pharmacia Biotech. Cat. # 17-0855-02) The hybirdization andwashing conditions were according to (J. Sambrook, E. F. Fritch and T.Maniatis (1989) A Laboratory Manaul Second. Ed. Vol. 1 pp. 2.69-2.81Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). OnePositive phage clone obtained form above screen was further purified.Southern analysis were carried out on digested phage DNA and a 9 kb SacI fragment hybirdized for this clone. This fragment was subcloned intopBlueScript KS vector, and further digested with EcoRI and southernanalyzed. A smaller 5 kb fragment hybirdized to the rat GPR14 probementioned above was subcloned into pBlueScript for sequence analysis,the sequence was determined by automated sequencer. A total of 2126 bpwere sequenced, this includes an open reading frame enconding a peptideof 390 residues. This sequence is highly homologus to Rat GPR14, asshown by sequence comparisons and also by fasta analysis against theGenbank nucleotid data base.

Example 2: Mammalian Cell Expression

The receptors of the present invention are expressed in either humanembryonic kidney 293 (HEK293) cells or adherent dhfr CHO cells. Tomaximize receptor expression, typically all 5' and 3' untranslatedregions (UTRs) are removed from the receptor cDNA prior to insertioninto a pCDN or pCDNA3 vector. The cells are transfected with individualreceptor cDNAs by lipofectin and selected in the presence of 400 mg/mlG418. After 3 weeks of selection, individual clones are picked andexpanded for further analysis. HEK293 or CHO cells transfected with thevector alone serve as negative controls. To isolate cell lines stablyexpressing the individual receptors, about 24 clones are typicallyselected and analyzed by Northern blot analysis. Receptor mRNAs aregenerally detectably in about 50% of the G418-resistant clones analyzed.

Example 3 Ligand Bank for Binding and Functional Assays

A bank of over 200 putative receptor ligands has been assembled forscreening. The bank comprises: transmitters, hormones and chemokinesknown to act via a human seven transmembrane (7TM) receptor; naturallyoccurring compounds which may be putative agonists for a human 7TMreceptor, non-mammalian, biologically active peptides for which amammalian counterpart has not yet been identified; and compounds notfound in nature, but which activate 7TM receptors with unknown naturalligands. This bank is used to initially screen the receptor for knownligands, using both functional (i.e. calcium, cAMP, microphysiometer,oocyte electrophysiology, etc, see below) as well as binding assays.

Example 4: Ligand Binding Assays

Ligand binding assays provide a direct method for ascertaining receptorpharmacology and are adaptable to a high throughput format. The purifiedligand for a receptor is radiolabeled to high specific activity (50-2000Ci/mmol) for binding studies. A determination is then made that theprocess of radiolabeling does not diminish the activity of the ligandtowards its receptor. Assay conditions for buffers, ions, pH and othermodulators such as nucleotides are optimized to establish a workablesignal to noise ratio for both membrane and whole cell receptor sources.For these assays, specific receptor binding is defined as totalassociated radioactivity minus the radioactivity measured in thepresence of an excess of unlabeled competing ligand. Where possible,more than one competing ligand is used to define residual nonspecificbinding.

Example 5: Functional Assay in Xenopus Oocytes

Capped RNA transcripts from linearized plasmid templates encoding thereceptor cDNAs of the invention are synthesized in vitro with RNApolymerases in accordance with standard procedures. In vitro transcriptsare suspended in water at a final concentration of 0.2 mg/ml. Ovarianlobes are removed from adult female toads, Stage V defolliculatedoocytes are obtained, and RNA transcripts (10 ng/oocyte) are injected ina 50 nl bolus using a microinjection apparatus. Two electrode voltageclamps are used to measure the currents from individual Xenopus oocytesin response to agonist exposure. Recordings are made in Ca2+ freeBarth's medium at room temperature. The Xenopus system can be used toscreen known ligands and tissue/cell extracts for activating ligands.

Example 6: Microphysiometric Assays

Activation of a wide variety of secondary messenger systems results inextrusion of small amounts of acid from a cell. The acid formed islargely as a result of the increased metabolic activity required to fuelthe intracellular signaling process. The pH changes in the mediasurrounding the cell are very small but are detectable by the CYTOSENSORmicrophysiometer (Molecular Devices Ltd., Menlo Park, Calif.). TheCYTOSENSOR is thus capable of detecting the activation of a receptorwhich is coupled to an energy utilizing intracellular signaling pathwaysuch as the G-protein coupled receptor of the present invention.

Example 7: Extract/Cell Supernatant Screening

A large number of mammalian receptors exist for which there remains, asyet, no cognate activating ligand (agonist). Thus, active ligands forthese receptors may not be included within the ligands banks asidentified to date. Accordingly, the 7TM receptor of the invention isalso functionally screened (using calcium, cAMP, microphysiometer,oocyte electrophysiology, etc., functional screens) against tissueextracts to identify natural ligands. Extracts that produce positivefunctional responses can be sequencially subfractionated until anactivating ligand is isolated identified.

Example 8: Calcium and cAMP Functional Assays

7TM receptors which are expressed in HEK 293 cells have been shown to becoupled functionally to activation of PLC and calcium mobilizationand/or cAMP stimuation or inhibition. Basal calcium levels in the HEK293 cells in receptor-transfected or vector control cells were observedto be in the normal, 100 nM to 200 nM, range. HEK 293 cells expressingrecombinant receptors are loaded with fura 2 and in a single day>150selected ligands or tissue/cell extracts are evaluated for agonistinduced calcium mobilization. Similarly, HEK 293 cells expressingrecombinant receptors are evaluated for the stimulation or inhibition ofcAMP production using standard cAMP quantitation assays. Agonistspresenting a calcium transient or cAMP flucuation are tested in vectorcontrol cells to determine if the response is unique to the transfectedcells expressing receptor.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2126 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GACAAAACTGGGTACGGGCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAAT60                TCGTTTCCCTGTATGAGAAATGGAGATTGCAGAGGCCTTCCTCTCCTTACATGTTCTTCT120               ATTTGGACTTTTAAAGTCAGTAGCTACTAGTTTTGCAATCTAAAGAAAACATTTTTTTAA180               ATGTACAAGTCAAATAAATACGAGAAAGGACTCAGGAGTAAGTGGGCCCCACCTGTGCAC240               AGACAAGAAAGTGAGGCCTGGGGGGGCGCACTGGGCAGAGCCCAGGACTCCCAGTTCTGT300               CCACTGCCGACCTCTGCCCCAGGGGCTGCCCTCCTGTGTTCCGGCTTTCAGAAAAGCCCA360               GTTCATCCCAGAGGCCATGGGACCTACAGTGAGGGGGGGGCAGGGGTCCTGCTGGGGCAT420               GCGGGGGTCGGGGAGGGGGGTTGGGGCAGCTCGTCTGGTGGCTCTTGAGTCCTCCTGCAG480               AGCTGGTGGCTTCCAGAGAGTCCCGAGAGTTGGAGGGCACTGGGGAGCCCACGTGACTCT540               GTGGGAACGAGGCCATCACAGTGGCCTCCTGGGAGCGGAAGGTGTTGCCTGATTTGCTTC600               TTTCCCCACAGGCTGAGCTGGTTGCCCACAGGGGCCCCCGCCCCATCTCAGGGAGTGTCC660               ACCCAGCCCTGAGCCCGTCGTGAGGGGTCAGAGATGGCGCTGACCCCCGAGTCCCCGAGC720               AGCTTCCCTGGGCTGGCCGCCACCGGCAGCTCTGTGCCGGAGCCGCCTGGCGGCCCCAAC780               GCAACCCTCAACAGCTCCTGGGCCAGCCCGACCGAGCCCAGCTCCCTGGAGGACCTGGTG840               GCCACGGGCACCATTGGGACTCTGCTGTCGGCCATGGGCGTGGTGGGCGTGGTGGGCAAC900               GCCTACACGCTGGTGGTCACCTGCCGCTCCCTGCGTGCGGTGGCCTCCATGTACGTCTAC960               GTGGTCAACCTGGCGCTGGCCGACCTGCTGTACCTGCTCAGCATCCCCTTCATCGTGGCC1020              ACCTACGTCACCAAGGAGTGGCACTTCGGGGACGTGGGCTGCCGCGTGCTCTTCGGCCTG1080              GACTTCCTGACCATGCACGCCAGCATCTTCACGCTGACCGTCATGAGCAGCGAGCGCTAC1140              GCTGCGGTGCTGCGGCCGCTGGACACCGTGCAGCGCCCCAAGGGCTACCGCAAGCTGCTG1200              GCGCTGGGCACCTGGCTGCTGGCGCTGCTGCTGACGCTGCCCGTGATGCTGGCCATGCGG1260              CTGGTGCGCCGGGGTCCCAAGAGCCTGTGCCTGCCCGCCTGGGGCCCGCGCGCCCACCGC1320              GCCTACCTGACGCTGCTCTTCGCCACCAGCATCGCGGGGCCCGGGCTGCTCATCGGGCTG1380              CTCTACGCGCGCCTGGCCCGCGCCTACCGCCGCTCGCAGCGCGCCTCCTTCAAGCGGGCC1440              CGGCGGCCGGGGGCGCGCGCGCTGCGCCTGGTGCTGGGCATCGTGCTGCTCTTCTGGGCC1500              TGCTTCCTGCCCTTCTGGCTGTGGCAGCTGCTCGCCCAGTACCACCAGGCCCCGCTGGCG1560              CCGCGGACGGCGCGCATCGTCAACTACCTGACCACCTGCCTCACCTACGGCAACAGCTGC1620              GCCAACCCCTTCCTCTACACGCTGCTCACCAGGAACTACCGCGACCACCTGCGCGGCCGC1680              GTGCGGGGCCCGGGCAGCGGGGGAGGCCGGGGGCCCGTTCCCTCCCTGCAGCCCCGCGCC1740              CGCTTCCAGCGCTGTTCGGGCCGCTCCCTGTCTTCCTGCAGCCCACAGCCCACTGACAGC1800              CTCGTGCTGGCCCCAGCGGCCCCGGCCCGACCTGCGCCCGAGGGTCCCAGGGCCCCGGCG1860              TGAGCACGCGGAGGGGCGGCTGGAGTCCAGGCGGGGACGCGCCCCAAAGCCCCAGCCACT1920              CCCGGGAGCCCCCCCAACTCCCAAATCACAGGCCCTGCCCCTCCTCCGTCCCCTTCTGGA1980              AAGATCCTGCTCGCTTCCCCTCAGCGCCCTTCCCGTGATGCCCAGAAGCGCCCACCCGCC2040              TCCCTGAGGGTCTCCAGGAGGCTCCAGCGCAGTCCCGGCTTCTGGAGACATGGCTTCGTC2100              ACAGAGGGCAGCAGGCGCCATTGCCC2126                                                (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 389 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAlaLeuThrProGluSerProSerSerPheProGlyLeuAlaAla                              151015                                                                        ThrGlySerSerValProGluProProGlyGlyProAsnAlaThrLeu                              202530                                                                        AsnSerSerTrpAlaSerProThrGluProSerSerLeuGluAspLeu                              354045                                                                        ValAlaThrGlyThrIleGlyThrLeuLeuSerAlaMetGlyValVal                              505560                                                                        GlyValValGlyAsnAlaTyrThrLeuValValThrCysArgSerLeu                              65707580                                                                      ArgAlaValAlaSerMetTyrValTyrValValAsnLeuAlaLeuAla                              859095                                                                        AspLeuLeuTyrLeuLeuSerIleProPheIleValAlaThrTyrVal                              100105110                                                                     ThrLysGluTrpHisPheGlyAspValGlyCysArgValLeuPheGly                              115120125                                                                     LeuAspPheLeuThrMetHisAlaSerIlePheThrLeuThrValMet                              130135140                                                                     SerSerGluArgTyrAlaAlaValLeuArgProLeuAspThrValGln                              145150155160                                                                  ArgProLysGlyTyrArgLysLeuLeuAlaLeuGlyThrTrpLeuLeu                              165170175                                                                     AlaLeuLeuLeuThrLeuProValMetLeuAlaMetArgLeuValArg                              180185190                                                                     ArgGlyProLysSerLeuCysLeuProAlaTrpGlyProArgAlaHis                              195200205                                                                     ArgAlaTyrLeuThrLeuLeuPheAlaThrSerIleAlaGlyProGly                              210215220                                                                     LeuLeuIleGlyLeuLeuTyrAlaArgLeuAlaArgAlaTyrArgArg                              225230235240                                                                  SerGlnArgAlaSerPheLysArgAlaArgArgProGlyAlaArgAla                              245250255                                                                     LeuArgLeuValLeuGlyIleValLeuLeuPheTrpAlaCysPheLeu                              260265270                                                                     ProPheTrpLeuTrpGlnLeuLeuAlaGlnTyrHisGlnAlaProLeu                              275280285                                                                     AlaProArgThrAlaArgIleValAsnTyrLeuThrThrCysLeuThr                              290295300                                                                     TyrGlyAsnSerCysAlaAsnProPheLeuTyrThrLeuLeuThrArg                              305310315320                                                                  AsnTyrArgAspHisLeuArgGlyArgValArgGlyProGlySerGly                              325330335                                                                     GlyGlyArgGlyProValProSerLeuGlnProArgAlaArgPheGln                              340345350                                                                     ArgCysSerGlyArgSerLeuSerSerCysSerProGlnProThrAsp                              355360365                                                                     SerLeuValLeuAlaProAlaAlaProAlaArgProAlaProGluGly                              370375380                                                                     ProArgAlaProAla                                                               385                                                                           __________________________________________________________________________

What is claimed is:
 1. An isolated polynucteotide comprising anucleotide sequence encoding the polypeptide having the amino acidsequence set forth in SEQ ID NO:2.
 2. The polynucleotide of claim 1wherein said nucleotide sequence comprises the entire coding region ofthe nucleotide sequence of SEQ ID NO: 1 encoding the polypeptide havingthe amino acid sequence set forth in SEQ ID NO:2.
 3. The polynucleotideof claim 1 comprising the nucleotide sequence set forth in SEQ ID NO: 1.4. An isolated polynucleotide comprising a nucleotide sequence encodinga naturally occurring human allelic variant of the polypeptide havingthe amino acid sequence set forth in SEQ ID NO:2.
 5. The polynucleotideof any one of claims 1, 2, 3 or 4 which is DNA.
 6. An isolatedpolynucleotide comprising an RNA molecule corresponding to thepolynucleotide of claim
 1. 7. The isolated polynucleotide of claim 1wherein said polynucleotide is an RNA transcript of the entire length ofSEQ ID NO:
 1. 8. The isolated polynucleotide of claim 1 wherein saidpolynucleotide is an RNA transcript of the entire coding region of SEQID NO:1.
 9. An isolated polynucleotide which is complementary to any oneof the isolated polynucleotides of claims 1, 2, 3, 4, 6, 7 and
 8. 10. Anexpression vector comprising a nucleic acid encoding the polypeptidehaving the amino acid sequence set forth in SEQ ID NO:2.
 11. A processfor producing a cell which produces the polypeptide having the aminoacid sequence set forth in SEQ ID NO:2 comprising transforming ortransfecting a host cell with the expression vector of claim 10 suchthat the host cell, under appropriate culture conditions, produces saidpolypeptide.
 12. A host cell produced by the process of claim
 11. 13. Anisolated host cell transfected or transformed with the nucleic acidencoding the polypeptide having the amino acid sequence set forth in SEQID NO:2.
 14. A process for producing the polypeptide having the aminoacid sequence set forth in SEQ ID NO:2 comprising culturing the hostcell of claim 13 under conditions sufficient for the production of saidpolypeptide and recovering said polypeptide from the culture.
 15. Anexpression vector comprising a nucleic acid encoding a naturallyoccurring human allelic variant of the polypeptide having the amino acidsequence set forth in SEQ ID NO:2.
 16. A process for producing a cellwhich produces a naturally occurring human allelic variant of thepolypeptide having the amino acid sequence set forth in SEQ ID NO:2comprising transforming or transfecting a host cell with the expressionvector of claim 15 such that the host cell, under appropriate cultureconditions, produces said variant.
 17. A host cell produced by theprocess of claim
 16. 18. An isolated host cell transfected ortransformed with the nucleic acid encoding a naturally occurring humanallelic variant of the polypeptide having the amino acid sequence setforth in SEQ ID NO:2.
 19. A process for producing a naturally occurringhuman allelic variant of the polypeptide having the amino acid sequenceset forth in SEQ ID NO:2 comprising culturing the host cell of claim 18under conditions sufficient for the production of said variant andrecovering said variant from the culture.